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| Status |
Public on May 29, 2024 |
| Title |
Pol_II_ChIPseq_GFP |
| Sample type |
SRA |
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| Source name |
dome
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| Organism |
Danio rerio |
| Characteristics |
tissue: dome
genotype: Wild type
treatment: GFP injection
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
100 embryos at dome stage were collected for each sample and fixed in cross-linking buffer (50mM HEPES pH8.0, 0.1 M NaCl, 1 mM EDTA, 0.5 M EGTA, 1 % Formaldehyde) for 15 min at room temperature. For chromatin fragmentation, 400 μl ChIP lysis buffer (50 mM Tris, pH 8.0, 10 mM EDTA, 0.1 % Sodium Deoxycholate, 1% Triton-X) with 1 × PIC and 1 mM PMSF were added to fixed embryos and incubated in ice for 20 min. Then, sonication was performed on ME220 with 10% duty factor, 75 peak incident power, 1000 cycles/burst, and for 600 second per tube. Fragmented chromatins were centrifuged at 12,000 rpm for 10 min at 4°C to remove cellular debris. For IP, 2 μg RNA Pol II antibody (Active Motif, no. 61667) and 10 μl Dynabeads™ Protein G were added to the fragmented chromatin solution and incubated at 4°C overnight with rotator at speed of 10 rpm. Beads containing immuno-bound chromatin were collected by placing the microfuge tube on a magnet rack and washed with ChIP lysis buffe 2 times, High salt wash buffer (10 mM Tris, pH 8.0, 500 mM NaCl, 1 mM EDTA, 1 % Triton-X, 0.1 % SDS, and 0.1% Sodium Deoxycholate) 2 times, LiCl wash buffer (10 mM Tris, pH 8.0, 250mM LiCl, 1 mM EDTA, 1 % Triton-X, 0.5 % SDS, and 0.5% Sodium Deoxycholate) 1 time, and TE 2 times, mixing 5 minutes for each wash on a rotator at 4°C. Then, the beads were suspended in 200 μl IP Elution Buffer with 200 μg/ml Proteinase K and incubated at 65°C overnight for reverse cross-linking, following DNA extraction with an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1, pH 7.8) with phase-lock tubes. The supernatant containing ChIP DNA was precipitated by addition of an equal volume of isopropanol and 1 μl GlycoBlue (Thermo Fisher) for 12 h at -20°C. Accel-NGS 1S Plus DNA Library Kit was used for library construction following the manual.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
For Pol II ChIP-seq data analysis, reads were aligned to the zebrafish danRer7 genome with Bowtie 2 to get alignment files (SAM). Then, SAM files were converted to BAM files following sorting and indexing with samtools (Li et al., 2009) (version 1.3). Duplicates were removed by Picard tools (http://broadinstitute.github.io/picard). For visualization, aligned reads were converted to normalized coverage files (bigWig) by using bamCoverage from deepTools (Ramirez et al., 2014) (version 2.4.2). To compare enrichment of Pol II signal in genes among samples, raw counts for the feature of genes were extracted by FeatureCounts (Liao et al., 2014) (version 2.0.3) and differential Pol II enriched genes were analyzed by using R package NOISeq (version 3.17) according to the user's guide (Tarazona et al., 2015).
bigWig
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| Submission date |
Jun 02, 2023 |
| Last update date |
May 29, 2024 |
| Contact name |
li kuan |
| E-mail(s) |
likuan@cibr.ac.cn
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| Organization name |
Tsinghua University
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| Street address |
zhongguancun
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| City |
Beijing |
| ZIP/Postal code |
100084 |
| Country |
China |
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| Platform ID |
GPL24995 |
| Series (1) |
| GSE183453 |
R-loop landscapes during parental-to-zygotic transition in zebrafish |
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| Relations |
| BioSample |
SAMN35572833 |
| SRA |
SRX20580010 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7444947_Pol_II_ChIPseq_GFP_RPGC.bw |
112.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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